Sample & Assay Technologies

Chromatin-Based Regulation of Gene Expression
George J. Quellhorst, Jr., PhD
Associate Direct...
Sample & Assay Technologies

Topics to be Discussed

Importance of Chromatin-Based Regulation
Mechanism & Pathway
Reading,...
Sample & Assay Technologies

Chromatin = DNA + DNA Binding Proteins
Structural (Histones) & Functional (Transcription Fact...
Sample & Assay Technologies

Transcriptional Regulation of Gene Expression
An Evolving Picture

TF Activating Signal
Trans...
Writing, Reading, Erasing DNA Methylation

Sample & Assay Technologies

NH 2
N

N

+ DNMT

O

O
AdoHcy

NH 2
N

- DNMT

CH...
Sample & Assay Technologies

DNA Methylation Dependent Transcriptional Repression
Possible Molecular Mechanisms

Singal R ...
Sample & Assay Technologies

Occurrence & Study of DNA Methylation

70% to 90% of CpG dinucleotides are methylated in heal...
Sample & Assay Technologies

The Histone Code

Acetylation
H3ac
Active Euchromatin
H3K9ac
Active Euchromatin
H4ac
Active E...
Sample & Assay Technologies

Writing, Reading, Erasing the Histone Code

Histone Lysine Acetylation
Neutralizes basic hist...
Sample & Assay Technologies

DNA Methylation & Histone Modification Interplay

Ikegami K, Ohgane J, Tanaka S, Yagi S, Shio...
Sample & Assay Technologies

DNA Methylation & Histone Modification Interplay

Kondo Y. (2009) Epigenetic cross-talk betwe...
Sample & Assay Technologies

Examples of Experimental Studies
Questions Asked & Methods Used

Question: Is expression of m...
Sample & Assay Technologies

Promoter Methylation Decreases Gene Expression

ERBB4 (HER4) in Breast Cancer Cell Lines
Why ...
Sample & Assay Technologies

Methylation Inhibits TF Binding but Recruits MeCP2

CREB, ATF2 and MeCP2 Binding to Insulin P...
Sample & Assay Technologies

Is Increased Gene Expression & TF Binding Epigenetic?
Ethanol & 5-aza-C Similarly Increase GR...
Sample & Assay Technologies

Removing Methylation Restores Gene Expression
Ethanol Treatment Decreases GRIN2B Methylation,...
Sample & Assay Technologies

Epigenetic Regulation Keeps Oncogenes Suppressed
MUC1 Expression & Regulation in Different Ca...
Sample & Assay Technologies

Both DNA Methylation & Histone Modification Play Roles
MUC1 Expression & Regulation in Differ...
Sample & Assay Technologies

Model for Epigenetic Regulation of Gene Expression

Unmethylated DNA + Acetylated H3 =
Strong...
Sample & Assay Technologies

SUMMARY

Epigenetic Regulation of Gene Expression Important to Research in …
Cancer & Toxicol...
Sample & Assay Technologies

How can you get started analyzing epigenetics?
QIAGEN Epigenetic Assay Technologies

DNA Meth...
Sample & Assay Technologies

Helping you get started …
More Technology-Focused Webinars

Profile the Methylation Status of...
Sample & Assay Technologies

Helping you get started …
With Your First or Next Order of Reagents
Get 20% any of the follow...
Sample & Assay Technologies

Q&A

Chromatin-Based Regulation of Gene Expression
George J. Quellhorst, Jr., PhD
Associate D...
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Chromatin regulation 2013

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Chromatin regulation 2013

  1. 1. Sample & Assay Technologies Chromatin-Based Regulation of Gene Expression George J. Quellhorst, Jr., PhD Associate Director, R&D, Biological Research Content Development
  2. 2. Sample & Assay Technologies Topics to be Discussed Importance of Chromatin-Based Regulation Mechanism & Pathway Reading, Writing, Erasing “the Code” Typical Questions Asked & Methods Used Examples from the Literature How QIAGEN can help Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 2
  3. 3. Sample & Assay Technologies Chromatin = DNA + DNA Binding Proteins Structural (Histones) & Functional (Transcription Factors) Matouk, C. C. et al. Circ Res 2008;102:873-887. Copyright © 2008 American Heart Association Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 3
  4. 4. Sample & Assay Technologies Transcriptional Regulation of Gene Expression An Evolving Picture TF Activating Signal Transduction Cascades THE Gene Expression Regulation Frontier! Highly Dynamic Promoter Context NFκB + p53 ? – SWI/SNF or INO80 or Mediator Complex RNA Pol II Structural Gene TSS (+1) mRNA or miRNA TBP p53 BS NFκB BS Distal Promoter Histones Me Me Me Me Me Me Me OAc OAc OAc Proximal Promoter Histone Modification DNA Methylation Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 4
  5. 5. Writing, Reading, Erasing DNA Methylation Sample & Assay Technologies NH 2 N N + DNMT O O AdoHcy NH 2 N - DNMT CH3 O C H3 N S -E n z N AdoMet H NH 2 H N R Cytosine 5-Methyl-Cytosine Modification of cytosine in CpG dinucleotide at 5-position with methyl (CH3) group Written by DNA Methyltransferases DNMT1: Maintenance Methylation DNMT3A & DNMT3B: De Novo Methylation Read by Transcription Factors Methyl DNA Binding Protein Histone Modification Enzymes Erased by … DNA Demethylases? 1 Normal 2 3 Expression 1 2 3 No Expression Promoter Region Tumor Suppressor Genes Cancer Herman JG, Baylin SB. (2003) Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med 20:2042. © 2003 Massachusetts Medical Society Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 5
  6. 6. Sample & Assay Technologies DNA Methylation Dependent Transcriptional Repression Possible Molecular Mechanisms Singal R and Ginder GD (1999) DNA Methylation. Blood 93:4059. © 1999 by The American Society of Hematology Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 6
  7. 7. Sample & Assay Technologies Occurrence & Study of DNA Methylation 70% to 90% of CpG dinucleotides are methylated in healthy somatic cells Representing 3% to 6% of all cytosines Less than expected frequency based on genomic GC content CpG Island = relatively low GC content but high CpG 7% of all CpG dinucleotides Associated with 5’ regulatory regions of 40-60% of human genes Typically unmethylated Cancer Genome-wide hypomethylation except CpG islands ~ X chromosome inactivation Development Cell-type specific methylation @ proximal promoter of affected genes 5-azacytidine (5-aza-C, DAC) Keeps CpG from being methylated turning gene expression back on Cancer treatment Mechanism of action experimental tool Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 7
  8. 8. Sample & Assay Technologies The Histone Code Acetylation H3ac Active Euchromatin H3K9ac Active Euchromatin H4ac Active Euchromatin Methylation H3K4me1 Non-TSS H3K4me2 Active Euchromatin H3K4me3 Active Euchromatin H3K9me1 Inactive Euchromatin H3K9me2 Heterochromatin H3K9me3 Heterochromatin H3K27me3 Inactive Euchromatin H3K36me3 Non-TSS H3K79me3 Active Euchromatin H4K20me3 Heterochromatin Phosphorylation & Ubiquitination Less clear . . . . . . . . . . . . . Kondo Y. (2009) Epigenetic cross-talk between DNA methylation and histone modifications in human cancers. Yonsei Med J. 50:455. © Yonsei University College of Medicine 2009 Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 8
  9. 9. Sample & Assay Technologies Writing, Reading, Erasing the Histone Code Histone Lysine Acetylation Neutralizes basic histone charge preventing higher order compaction More open and accessible for transcription Written by Histone Acetyltransferases (HATs) Read by Bromodomain proteins – Chromatin Remodeling Factors Erased by Histone Deacetylases (HDACs) Trichostatin A (TSA) inhibits most HDACs – Class I (HDAC1-3, HDAC8) & Class II (HDAC4-7, HDAC9-10) – Allows histones to be re-acetylated turning gene expression back on – Cancer treatment – Mechanism of action experimental tool Histone Lysine Methylation Closed form, inactive for transcription Written by Trithorax Group & SET domain proteins Read by ING family & Chromobox homolog proteins – Chromatin Remodeling Factors Erased by Jumonji Domain (JARID/KMD) Lysine Demethylases Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 9
  10. 10. Sample & Assay Technologies DNA Methylation & Histone Modification Interplay Ikegami K, Ohgane J, Tanaka S, Yagi S, Shiota K. (2009) Interplay between DNA methylation, histone modification and chromatin remodeling in stem cells and during development. Int J Dev Biol. 2009;53(2-3):203-14. © 2009 UBC Press Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 10
  11. 11. Sample & Assay Technologies DNA Methylation & Histone Modification Interplay Kondo Y. (2009) Epigenetic cross-talk between DNA methylation and histone modifications in human cancers. Yonsei Med J. 50:455. © Yonsei University College of Medicine 2009 Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 11
  12. 12. Sample & Assay Technologies Examples of Experimental Studies Questions Asked & Methods Used Question: Is expression of my gene of interest (GOI) regulated by epigenetics? Is its promoter methylated? What histone modifications localize at its transcription start site? Do these epigenetic marks interfere with transcription factor (TF) binding? Can its silenced expression be turned on again and TF binding be restored by erasing epigenetic marks? Methods: DNA Methylation Analysis: Bisulfite Treatment (Converts C but not Me-C to U) followed by … – Microarray, Sequencing – Methyl-Specific PCR – primers without Cs except at 3’-end Histone Modification and Transcription Factor (TF) Binding Analysis Chromatin Immunoprecipitation (ChIP) followed by PCR – Anti-histone modification or anti-TF pull down & DNA purification – PCR primers to amplify promoter region for GOI Erase epigenetic marks with 5-aza-C and/or TSA Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 12
  13. 13. Sample & Assay Technologies Promoter Methylation Decreases Gene Expression ERBB4 (HER4) in Breast Cancer Cell Lines Why are some breast cancers HER4 positive and some HER4 negative? RT-PCR: HER4 Bisulfite Pyrosequencing Results More Methylation Less Expression Das PM, Thor AD, Edgerton SM, Barry SK, Chen DF, Jones FE. (2010) Reactivation of epigenetically silenced HER4/ERBB4 results in apoptosis of breast tumor cells. Oncogene 29:5214. © 2010 Macmillan Publishers Limited Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 13
  14. 14. Sample & Assay Technologies Methylation Inhibits TF Binding but Recruits MeCP2 CREB, ATF2 and MeCP2 Binding to Insulin Promoter What are the consequences of tissue-specific methylation of the insulin promoter? Chromatin Immunoprecipitation followed by PCR from NIT-1 mouse insulinoma cell line IP: Various TFs PCR: Endogenous Promoter IP: Various TFs PCR: Transfected Reporter Plasmids Kuroda A et al. (2009) Insulin gene expression is regulated by DNA methylation. PLoS One. 4:e6953. © 2009 Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 14
  15. 15. Sample & Assay Technologies Is Increased Gene Expression & TF Binding Epigenetic? Ethanol & 5-aza-C Similarly Increase GRIN2B Expression & TF Binding RT-PCR: GRIN2B CIE: Chronic Intermittent Ethanol CIEW2: CIE + 2-day withdrawal CIEW5: CIE + 5-day withdrawal 5’AZA: 5-Azacytidine Why does ethanol activate expression of GRIN2B? Chromatin Iummunoprecipitation IP: Various TFs PCR: GRIN2B Promoter TFBS Primary cortical cultured neurons Qiang M, Denny A, Chen J, Ticku MK, Yan B, Henderson G. (2010) The site specific demethylation in the 5'-regulatory area of NMDA receptor 2B subunit gene associated with CIE-induced up-regulation of transcription. PLoS One 20:e8798. © 2010 Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 15
  16. 16. Sample & Assay Technologies Removing Methylation Restores Gene Expression Ethanol Treatment Decreases GRIN2B Methylation, DNMT1 Expression Why does ethanol activate expression of GRIN2B? SAM = S-adenosyl-L-methionine, DNMT substrate – Rescues hypomethylation RT-PCR: DNMT1 Bisulfite Pyrosequencing Results CIE: Chronic Intermittent Ethanol CIEW2: CIE + 2-day withdrawal CIEW5: CIE + 5-day withdrawal 5’AZA: 5-Azacytidine Qiang M, Denny A, Chen J, Ticku MK, Yan B, Henderson G. (2010) The site specific demethylation in the 5'-regulatory area of NMDA receptor 2B subunit gene associated with CIE-induced up-regulation of transcription. PLoS One 20:e8798. © 2010 Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 16
  17. 17. Sample & Assay Technologies Epigenetic Regulation Keeps Oncogenes Suppressed MUC1 Expression & Regulation in Different Cancer Cell Lines Why do some cancers NOT express MUC1, a potential marker for malignancy? Cell Line PANC1 MDA-MB-453 Caco2 LS147T MUC1 Expression Restored By … Demethylation but not histone re-acetylation Demethylation and histone re-acetylation, but not synergistically Demethylation but not histone re-acetylation Histone re-acetylation, somewhat, perhaps synergy demethylation Yamada N et al. (2008) MUC1 expression is regulated by DNA methylation and histone H3 lysine 9 modification in cancer cells. Cancer Res 15:2708. © 2008 American Association for Cancer Research. Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 17
  18. 18. Sample & Assay Technologies Both DNA Methylation & Histone Modification Play Roles MUC1 Expression & Regulation in Different Cancer Cell Lines Do MUC1 Methylation Status and Histone Code Correlate with Gene Expression? Cell Line PANC1 LS147T MDA-MB-453 Caco2 Methylated? Y N Y Y Histone Marks (Inactive Euchromatin) (Heterochromatin) Heterochromatin Heterochromatin Yamada N et al. (2008) MUC1 expression is regulated by DNA methylation and histone H3 lysine 9 modification in cancer cells. Cancer Res 15:2708. © 2008 American Association for Cancer Research. Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 18
  19. 19. Sample & Assay Technologies Model for Epigenetic Regulation of Gene Expression Unmethylated DNA + Acetylated H3 = Strong Expression Methylated DNA + Acetylated H3 = Modest Expression Unmethylated DNA + Methylated H3 = Modest Expression Methylated DNA + Methylated H3 = Very Low Expression Yamada N et al. (2008) MUC1 expression is regulated by DNA methylation and histone H3 lysine 9 modification in cancer cells. Cancer Res 15:2708. © 2008 American Association for Cancer Research. Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 19
  20. 20. Sample & Assay Technologies SUMMARY Epigenetic Regulation of Gene Expression Important to Research in … Cancer & Toxicology Tissue-Specific Expression & Development Your research field “The Code” DNA methylation inhibits expression; DNA demethylation re-activates Histone acetylation permits while histone methylation suppresses expression DNA methylation & histone modification statuses both contribute & synergize Methods & Reagents Available & Well-Established DNA Methylation Analysis Pyrosequencing, Real-Time PCR Reverse methylation with 5-aza-C In vivo DNA-Protein Interactions Analysis Chromatin Immunoprecipitation (histone modifications or TFs) Real-Time PCR Re-acetylate histones with HDAC inhibitor TSA Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 20
  21. 21. Sample & Assay Technologies How can you get started analyzing epigenetics? QIAGEN Epigenetic Assay Technologies DNA Methylation Analysis of Gene or Pathway of Interest Pyrosequencing QIAGEN PyroMark CpG Assays Real-Time PCR QIAGEN EpiTect Methyl II PCR Assays & Arrays Chromatin Immunoprecipitation (ChIP) for Histone Marks & TF Binding Real-Time PCR QIAGEN EpiTect ChIP PCR Assays & Arrays Knock Down Expression of Histone Modification Enzymes FlexiTube siRNA & FlexiPlate siRNA SureSilencing shRNA Plasmids Analyze Expression of Gene or Pathway of Interest RT2 Profiler PCR Arrays Epigenetic Chromatin Modification Enzymes Epigenetic Chromatin Remodeling Factors Nearly 150 application, disease, pathway-focused arrays RT2 qPCR Primer Assays & QuantiTect Primer Assays Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 21
  22. 22. Sample & Assay Technologies Helping you get started … More Technology-Focused Webinars Profile the Methylation Status of Multiple Genes Simultaneously Without Bisulfite Tuesday, April 23, 2013 at 9:30 AM Eastern US Chromatin Immunoprecipitation and Real-Time PCR Technology Overview Wednesday, May 8, 2013 at 1:00 PM Eastern US PCR Arrays: The Real Pioneer in Real-Time PCR Analysis of Biological Pathways Monday, April 22, 2013 at 9:30 AM Eastern US Knock Down Your Favorite Genes or Pathways with Ease and Confidence Friday, April 19, 2013 at 1:00 PM Eastern US Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 22
  23. 23. Sample & Assay Technologies Helping you get started … With Your First or Next Order of Reagents Get 20% any of the following products: EpiTect Methyl II PCR Assays & Arrays EpiTect ChIP PCR Assays & Arrays SureSilencing shRNA Plasmids RT2 Profiler PCR Arrays RT2 qPCR Primer Assays PLUS Auto Emergency Kit! Simply refer to PROMO CODE: FDK-WN20W22 Only available in US & Canada; Expires April 30, 2013 More Resources to Help You Technical Support Email: support@sabiosciences.com Webinar Related Questions Email: qiawebinars@qiagen.com Also, please remember to complete our survey after the webinar. Chromatin-Based Regulation of Gene Expression, Webinar, Wednesday, April 17, 2013 23
  24. 24. Sample & Assay Technologies Q&A Chromatin-Based Regulation of Gene Expression George J. Quellhorst, Jr., PhD Associate Director, R&D, Biological Research Content Development

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